CN113874715A

CN113874715A - In vitro method for evaluating hemostatic agents in capillary models

Info

Publication number: CN113874715A
Application number: CN201980096772.0A
Authority: CN
Inventors: 许少鹏; 陈岱琳
Original assignee: Colgate Palmolive Co
Current assignee: Colgate Palmolive Co
Priority date: 2019-05-31
Filing date: 2019-05-31
Publication date: 2021-12-31
Also published as: WO2020242502A1; EP3977107A1

Abstract

An in vitro platelet assessment method of determining platelet aggregation area and platelet rolling velocity, the method comprising: generating a platelet suspension sample; producing a test matrix (129), wherein the test matrix (129) comprises collagen; setting up an evaluation system (10), including placing a test substrate (129) into the evaluation system (10); flowing a platelet suspension sample through an evaluation system (10); regulating the flow of the platelet suspension sample through the evaluation system (10); stopping the flow of the platelet suspension sample through the evaluation system (10); generating a test platelet suspension; flowing a test platelet suspension through an evaluation system (10); regulating flow of the test platelet suspension through the evaluation system (10); and stopping the flow of the platelet suspension through the evaluation system (10). Preferably, the evaluation system (10) comprises a medium source (150), a parallel plate flow chamber (100), a flow pump (200,300), a microscope (400), a camera (400) and a computer (600).

Description

In vitro method for evaluating hemostatic agents in capillary models

Background

Gingival bleeding is associated with many common oral conditions such as gingivitis. Gingival bleeding can be caused by the accumulation of plaque, the formation of a soft, sticky, colorless film of bacteria on the teeth and gums, and the production of toxins that can inflame or infect the gingival tissue, causing gingivitis. Gingivitis is the initial stage of gum disease and, if left untreated, can lead to periodontitis.

Various clinical evaluation methods have been developed to evaluate the efficacy of oral care compositions on gingival bleeding, such as gingival bleeding index, gingival sulcus bleeding index, and papillary bleeding score, among others. However, there have been no preclinical and/or in vitro tests for evaluating the efficacy of oral care compositions for gingival bleeding or as a hemostatic agent.

Accordingly, it would be useful to develop systems and methods for preclinical and/or in vitro assessment of oral care compositions or active ingredients for gingival bleeding and/or as hemostatic agents. And in particular to in vitro systems and methods for assessing the efficacy of an oral care composition or active ingredient as a hemostatic agent in a capillary model.

Disclosure of Invention

This summary is intended only to introduce a simplified summary of some aspects of one or more embodiments of the disclosure. Further areas of applicability of the present invention will become apparent from the detailed description provided hereinafter. This summary is not an extensive overview, is not intended to identify key or critical elements of the teachings or to delineate the scope of the disclosure. Rather, its sole purpose is to present one or more concepts in a simplified form as a prelude to the more detailed description that is presented later.

The foregoing and/or other aspects and utilities embodied in the present disclosure may be achieved by providing an in vitro platelet assessment method comprising generating a platelet suspension sample; generating a test matrix, wherein the test matrix comprises collagen; setting up an evaluation system comprising placing the test substrate into the evaluation system; flowing the platelet suspension sample through the evaluation system; adjusting the flow of the platelet suspension sample through the evaluation system; stopping the flow of the platelet suspension sample through the evaluation system; generating a test platelet suspension; flowing the test platelet suspension through the evaluation system; adjusting the flow of the test platelet suspension through the evaluation system; and stopping the flow of the test platelet suspension through the evaluation system.

The evaluation system may include: a medium source that holds at least one of a platelet suspension sample and a test platelet suspension; a flow chamber that receives at least one of the platelet suspension sample and the test platelet suspension from the media source; a flow pump that facilitates flow of at least one of the platelet suspension sample and the test platelet suspension through the flow chamber; a microscope configured to evaluate at least one of the platelet suspension sample and the test platelet suspension in the flow chamber; and a camera and computer operably connected to the microscope and configured to record images through the microscope and facilitate evaluation of at least one of the platelet suspension sample and the test platelet suspension in the flow chamber.

The flow chamber may be a parallel plate flow chamber comprising: a bottom plate configured to control flow of at least one of the platelet suspension sample and the test platelet suspension through the flow chamber; a sled configured to receive at least one of the platelet suspension sample and the test platelet suspension from the media source; and a gasket configured to control a height of a flow path in the flow chamber, wherein the bottom plate comprises a flow inlet configured to connect to the media source, a flow outlet configured to connect to the flow pump, and a vacuum port configured to connect to a vacuum pump.

Adjusting the flow of the platelet suspension sample through the evaluation system can comprise adjusting a flow rate of the platelet suspension sample that simulates the flow through a capillary bleeding model of the evaluation system; and the method may further comprise recording the behavior of platelets in the platelet suspension sample in the assessment system.

The flow rate simulated through the capillary bleeding model of the evaluation system may be about 0.04 mL/min.

The behaviour of the platelets in the platelet suspension sample can be recorded at 50fps/s for 2 minutes.

The behavior of platelets in the platelet suspension sample recorded in the evaluation system can be used to determine the platelet aggregation area and the platelet rolling velocity of the platelet suspension sample.

Generating a test platelet suspension can comprise adding a sample of an oral care composition or active agent to the platelet suspension sample to generate a test platelet suspension.

Adjusting the flow of the test platelet suspension through the evaluation system can include adjusting a flow rate of the test platelet suspension that simulates the flow through a capillary bleeding model of the evaluation system; and the method may further comprise recording the behavior of platelets in the test platelet suspension in the evaluation system.

The behaviour of the platelets in the test platelet suspension can be recorded at 50fps/s for 2 minutes.

Recording the behavior of platelets in the test platelet suspension by the evaluation system can be used to determine the platelet aggregation area and platelet rolling velocity of the test platelet suspension.

The foregoing and/or other aspects and utilities embodied in the present disclosure may also be achieved by providing an in vitro platelet assessment method comprising generating a platelet suspension sample; generating a test platelet suspension by adding a test agent to the platelet suspension sample; flowing the platelet suspension sample through an evaluation system and recording the behavior of platelets in the platelet suspension sample in the evaluation system; flowing the test platelet suspension through the evaluation system and recording the behavior of platelets in the test platelet suspension in the evaluation system; comparing the behavior of platelets in the platelet suspension sample in the evaluation system with the behavior of platelets in the test platelet suspension in the evaluation system to determine the hemostatic efficacy of the test agent; wherein at least one of flowing the platelet suspension sample through the evaluation system and flowing the test platelet suspension through the evaluation system comprises adjusting a flow rate that simulates flow through a capillary bleeding model of the evaluation system, and wherein comparing the behavior of the platelets comprises comparing a mean platelet aggregation area and a mean platelet rolling velocity of the platelet suspension sample to the test platelet suspension.

Drawings

The accompanying drawings, which are incorporated in and constitute a part of this specification, illustrate embodiments of the present teachings and together with the description, serve to explain the principles of the disclosure. In the drawings:

FIG. 1 shows an in vitro platelet evaluation system according to embodiments of the present disclosure

Fig. 2 illustrates an in vitro platelet evaluation method according to embodiments of the present disclosure.

Fig. 3-4 are photographs showing the difference between individual platelets and aggregated platelets on a collagen matrix.

It should be noted that some of the details of the drawings have been simplified and drawn to facilitate understanding of the teachings of the present invention rather than to maintain strict structural accuracy, detail, and scale.

Detailed Description

Reference will now be made in detail to exemplary embodiments of the present teachings, examples of which are illustrated in the accompanying drawings. Generally, the same reference numbers will be used throughout the drawings to refer to the same or like parts.

The following description of embodiments is provided to provide a more thorough understanding of the components, methods, compositions, and apparatus disclosed herein. Any examples given are intended to be illustrative and not limiting. It will be apparent, however, to one skilled in the art that the present invention may be practiced without these specific details. In other instances, well-known methods, procedures, and components have not been described in detail so as not to unnecessarily obscure aspects of the embodiments.

Throughout the specification and claims, the following terms take the meanings explicitly associated herein, unless the context clearly dictates otherwise. As used herein, phrases such as "in one embodiment," "in certain embodiments," and "in some embodiments" do not necessarily refer to the same embodiment, although they may be. Furthermore, as used herein, the phrases "in another embodiment" and "in some other embodiments" do not necessarily refer to a different embodiment, although they may be different. As described below, the various embodiments may be readily combined without departing from the scope or spirit of the present disclosure.

As used herein, the term "or" is an inclusive operator and is equivalent to the term "and/or" unless the context clearly dictates otherwise. Unless the context clearly dictates otherwise, the term "based on" is not exclusive and allows for being based on additional factors not described. In the specification, a recitation of "at least one of A, B and C" includes embodiments that include: A. b or C; A. multiple instances of B or C; or a combination of A/B, A/C, B/C, A/B/B/B/B/C, A/B/C, etc. In addition, throughout the specification, the meaning of "a/an" and "the" includes a plurality of references. The meaning of "in … …" includes "in … …" and "on … …".

It will also be understood that, although the terms first, second, etc. may be used herein to describe various elements, these elements should not be limited by these terms. These terms are only used to distinguish one element from another. For example, a first object, component, or step may be termed a second object, component, or step, and, similarly, a second object, component, or step may be termed a first object, component, or step, without departing from the scope of the present invention. A first object, component or step and a second object, component or step are each an object, component or step, respectively, but they should not be considered the same object, component or step. It will be further understood that the terms "comprises" and/or "comprising," when used in this specification, specify the presence of stated features, steps, operations, elements, and/or components, but do not preclude the presence or addition of one or more other features, steps, operations, elements, components, and/or groups thereof. Further, as used herein, the term "if" may be understood to mean "when … …" or "at … …" or "in response to a determination of … …" or "in response to a detection of … …", depending on the context.

Unless otherwise indicated, all physical properties defined below are measured at 20 ℃ to 25 ℃.

When referring to any numerical range herein, such ranges are understood to include each and every number and/or fraction between the stated range minimum and maximum and the endpoints. For example, a range of 0.5-6% would expressly include all intermediate values, e.g., 0.6%, 0.7%, and 0.9%, up to and including 5.95%, 5.97%, and 5.99%, etc. This applies equally to each and every other numerical characteristic and/or element range set forth herein, unless the context clearly dictates otherwise.

Moreover, all numerical values are "about" or "approximately" the stated value, and take into account experimental error and deviation as would be expected by one of ordinary skill in the art. It is to be understood that all values and ranges disclosed herein are approximate values and ranges, regardless of whether "about" is used in connection therewith.

Unless otherwise indicated, all percentages and amounts expressed herein and elsewhere in this specification are to be understood as referring to weight percentages. The amounts given are based on the effective weight of the material.

With regard to procedures, methods, techniques, and workflows according to some embodiments, some operations in the procedures, methods, techniques, and workflows disclosed herein may be combined and/or the order of some operations may be altered.

As used herein, "gingival bleeding" or "hemorrhage" refers to the leakage of blood from a damaged blood vessel in the circulatory system. For example, as described above, gingival bleeding may be due to leakage of blood from damaged capillaries in the gingiva surrounding the teeth, and may be a symptom of gingivitis or periodontal disease.

Platelets are a component of blood that responds to bleeding caused by vascular injury by clotting and initiating a blood clot. That is, platelet aggregation is the first step in blood coagulation, which involves platelet adhesion and the formation of soft aggregate emboli.

The effectiveness of an oral care composition or active ingredient to prevent, reduce or stop gum bleeding can be measured by the platelet aggregation area and/or the platelet rolling velocity. For example, anti-gingival bleeding or hemostatic efficacy may be demonstrated by a higher platelet aggregation area and/or a lower platelet rolling velocity.

The present inventors have developed novel in vitro platelet evaluation systems and methods for evaluating the efficacy of oral care compositions or active ingredients on gingival bleeding or as hemostatic agents. In particular, the novel flow cell system is designed to assess the growth and stability of platelet aggregation in the presence of a particular oral care composition or active ingredient. In some embodiments, the flow cell system is configured to represent a capillary bleeding model and can be used to measure the effect of an oral care composition or active ingredient on the platelet aggregation area and/or the platelet rolling velocity.

Fig. 1 shows an in vitro platelet evaluation system according to embodiments of the present disclosure. As shown in fig. 1, the in vitro platelet evaluation system 10 includes a flow chamber 100, a media source 150, a vacuum pump 200, a flow pump 300, a microscope 400, a camera 500, and a computer 600.

The flow chamber 100 can be implemented as a parallel plate flow chamber 100. For example, the Flow Chamber 100 can be a circular parallel plate Flow Chamber 100 comprising a base plate 110, a slide plate 120, and a gasket 130 (e.g., a GlycoTech Flow Chamber 31-001 circular parallel plate Flow Chamber commercially available from GlycoTech Corporation, USA).

The base plate 110 may include a flow inlet 111 and a flow outlet 112 through which a test medium may be poured. The base plate 110 may also include a vacuum port 113 through which vacuum may be applied. In some embodiments, the vacuum is configured to hold the base plate 110, gasket 130, and slide plate 120 together and help maintain a uniform channel height. In some embodiments, the test medium may comprise a platelet suspension sample and/or a test platelet suspension comprising a suitable amount of an oral care composition or active ingredient sample for evaluation.

The sled 120 may be configured to hold a test substrate 129. For example, the slide plate 120 may be implemented as a 35mm petri dish (which may be made of glass, for example) and configured to hold a collagen matrix.

The gasket 130 is configured to control the height of the flow path of the flow chamber 100. For example, the width or thickness of the gasket 130 may be selected to control the gap height of the flow chamber 100. That is, the gasket thickness can control the channel height. Gasket 130 may be selected based on the desired flow cell type, test sample, and sidewall shear stress.

In one embodiment, the channel height may be 0.127mm, the width may be 2.5mm, and the length may be 5 mm. The flow chamber can be configured to provide parallel plate dynamic flow with stable viscosity.

The media source 150 is configured to hold a test media to be flowed through the flow cell 100. A media source 150 may be connected to the flow inlet 111 to provide test media to the flow cell 100. The test medium may comprise a platelet suspension sample that is tested as a control and/or a test platelet suspension that is evaluated as a hemostatic agent, the test platelet suspension comprising a suitable amount of an oral care composition or active ingredient. In some embodiments, the flow chamber 100 is maintained at 37 ℃.

The vacuum pump 200 is connected to the vacuum port 113 and is configured to maintain a vacuum environment in the flow chamber 100. In some embodiments, the vacuum pump 200 also helps maintain a stable flow rate of the in vitro platelet evaluation system 10. For example, the vacuum pump 200 may be configured to evacuate air and fluidly connect the extracorporeal platelet evaluation system 10 to create a steady flow.

The flow pump 300 is configured to provide dynamic flow to the system. For example, the flow pump 300 may be implemented as an injection flow pump 300 connected to the flow outlet 110. An example of a syringe-type flow pump 300 is commercially available, such as the Harvard PHD 22/2000 syringe pump available from Harvard Apparatus of Holliston, MA.

The flow cell 100 can be placed within a microscope 400 (e.g., Axio Observer a1, commercially available from Zeiss, germany) that is connected to a high-speed camera 500 (e.g., Mikrotron GmbH MC 1310; commercially available from Norpix inc. The microscope 400 and camera 500 may be connected to a computer 600 to facilitate viewing and recording of media as it flows and/or is deposited in the flow cell 100. For example, computer 600 may contain image analysis software for automated image analysis.

Fig. 2 illustrates an in vitro platelet evaluation method according to embodiments of the present disclosure. The in vitro platelet evaluation method 800 may be described with reference to the in vitro platelet evaluation system 10 of fig. 1.

The method 800 may begin by generating a platelet suspension sample in operation 810. For example, a sample of fresh blood can be centrifuged and/or aspirated to separate platelets from the blood, which can then be resuspended in a neutral solution, such as PBS, to produce a platelet suspension sample. In some embodiments, each platelet suspension sample contains a predetermined number of platelets. In some embodiments, the platelet suspension sample may also serve as a control sample.

In operation 820, a test matrix may be prepared. For example, the collagen matrix may be placed on the sled 120 and incubated to prepare the collagen matrix for interaction with the platelet suspension. While not being bound by any particular theory, the inventors believe that during clotting, platelets adhere to each other and to collagen to form platelet emboli. Thus, after infusion of the platelet solution into the flow cell 100, the platelets can interact with the collagen and bind to each other on the collagen matrix in the sled 120. An effective coagulant may not only help platelets bind to the collagen matrix in the flow chamber 100 to slow its rolling speed, but may also enhance stable platelet-platelet adhesion, which brings more aggregated platelets to the sample, thereby increasing the total platelet aggregation area.

In operation 830, an evaluation system is set up. For example, the in vitro platelet evaluation system 10 may be set up: the slide plate 120 (with the collagen matrix placed therein) can be brought together with the gasket 120 and the base plate 110 to form the flow chamber 100. The platelet suspension sample can be placed within the media source 150 and the media source 150 can be connected to the flow inlet 111 of the flow cell 100. Similarly, the flow outlet 112 may be connected to an injection flow pump 300 and the vacuum port 113 may be connected to a vacuum pump 200. The flow cell 100 may then be placed within a microscope 400 and/or camera 500 (connected to a computer 600) configured to observe, record and measure the behavior of a platelet suspension sample within the in vitro platelet evaluation system 10.

In operation 840, a platelet suspension sample is flowed into flow chamber 110. For example, flow chamber 100 may be infused with a platelet suspension sample from media source 150, and syringe flow pump 300 may be configured to deliver 0.04mL/min (0.1 dyn/cm)²Flow stress) creates a flow of the platelet suspension sample through the flow cell 100. However, a higher flow rate may be used to initially load the platelet suspension sample into the flow cell 110. The platelet suspension sample may initially flow through the flow cell 100 for 2 minutes. In some embodiments, the flow rate in operation 840 can be between 0 and 0.5dyn/cm²Within the range of (1). In some embodiments, flow chamber 110 is maintained at room temperature (25 ℃).

Using a flow rate of 0.04mL/min will result in 0.1dyn/cm according to the following volumetric flow rate equation²Flow stress of (2):

(1)

wherein tau is_wIs dyn/cm²Mu is the apparent viscosity of the medium (e.g., H at 37 ℃ C.) in terms of side wall shear stress₂O ═ 0.0076P), Q is the volumetric flow rate in l/sec, "a" is the channel height (gasket thickness in cm), and "b" is the channel width (gasket width in cm).

In operation 850, the flow rate is adjusted and platelet behavior is recorded. For example, the syringe flow pump 300 may be configured to regulate (or maintain) the flow of the platelet suspension sample through the flow chamber 100 to 0.04 mL/min. In some embodiments, 0.04mL/min (0.1 dyn/cm)²) The flow rate mimics the blood shear stress in the oral capillaries. In other embodiments, the flow rate is adjusted to less than 0.2dyn/cm²To simulate a capillary bleeding model. In yet other embodiments, the flow rate is adjusted to simulate a capillary bleeding model.

The platelet suspension sample may flow through the flow cell 100 for an additional 3 minutes, and the camera 500 may be set to record 2 minutes of platelet behavior at high speed (50 fps/s). In some embodiments, high speed recording may be used to calculate the platelet rolling speed and/or platelet aggregation area of the sample. A 2 minute length can be used to simulate the average brushing time and thus the period of exposure to the oral care composition or active ingredient being evaluated. However, other video lengths from less than 1 minute to 2.5 minutes and up to 90 minutes may be used depending on the expected exposure period of the oral care composition or active ingredient being evaluated as a hemostatic agent.

The platelet rolling speed of the platelet suspension sample can then be measured. For example, the rolling distance of each platelet in the platelet suspension sample can be tracked in the high-speed recording described above for 30 seconds (from the 90 second point to the 120 second point). Then, the rolling speed of the platelet suspension sample was obtained by averaging all the obtained rolling speeds. To facilitate measurement of platelet rolling velocity, in some embodiments, only platelets with a particular mean diameter will be tracked and measured. For example, only platelets with a mean diameter of 2.557+/-0.929 μm were followed to measure their rolling speed. In other embodiments, a group of multiple platelets is selected for tracking. For example, 50 platelets with the desired average diameter may be tracked to determine the platelet rolling speed of the sample.

In some embodiments, image analysis may be performed on the high-speed recordings using the computer 600 to facilitate measurement of the platelet rolling speed and/or the platelet aggregation area of the platelet suspension sample. For example, platelet rolling distance may be captured by a camera 500 operatively connected to the microscope 400, and the rolling speed over a particular period of time may be measured by a computer 600 operatively connected to both the camera 500 and the microscope 400 using imaging software (e.g., Image Pro Plus version 6.0, Media Cybernetics).

In operation 860, the platelet suspension sample flow is stopped and the remaining volume is measured. For example, the syringe flow pump 300 may be configured to stop the flow of the platelet suspension sample through the flow cell 100. Then, the remaining volume of platelets and/or the platelet aggregation area on the collagen matrix was observed. For example, the sum of the areas of adhered platelets on the collagen matrix can be measured at the 120 second point of the high speed recording described above.

For example, the area of platelet aggregation may be captured by a camera 500 operatively connected to the microscope 400, and the area of platelet aggregation over a particular period of time may be measured by a computer 600 operatively connected to both the camera 500 and the microscope 400 using imaging software (e.g., Image Pro Plus version 6.0, Media Cybernetics). In some embodiments, a representative sample is used to calculate the average platelet aggregation area. For example, the computer may be configured to recognize only between 2-18 μm²Platelets within the range, i.e., over the conventional range of platelet areas, and 200 platelet cells were measured during each time period.

Fig. 3-4 are photographs showing the difference between individual platelets and aggregated platelets on a collagen matrix, as identified by a computer 600 operatively connected to both the camera 500 and the microscope 400.

In operation 870, a test platelet suspension is generated. For example, an appropriate concentration of the oral care composition or active ingredient to be evaluated is added to the platelet suspension sample in the media source 150 to produce a test platelet suspension. In one embodiment, 10ul of an oral composition sample may be added to 1000ul of a platelet suspension sample, i.e., in a ratio of 1: 100.

In operation 880, the test platelet suspension is flowed into the flow chamber. For example, the flow chamber 100 may infuse a test platelet suspension from the media source 150, and the syringe flow pump 300 may be configured to generate a flow of the test platelet suspension through the flow chamber 100 at 0.32 mL/min. The platelet suspension may initially flow through the flow chamber 100 for 4 minutes.

In operation 890, the flow rate is varied and platelet behavior is recorded. For example, the syringe flow pump 300 may be configured to regulate the flow of the test platelet suspension to simulate a capillary bleeding model, such as 0.04mL/min, through the flow chamber 100. The test platelet suspension may flow through the flow cell 100 for an additional 3 minutes, and the camera 500 may be set to record platelet behavior at high speed (50fps/s) for 2 minutes.

Similar to above, the platelet rolling speed of the test platelet suspension can then be measured by tracking the rolling distance of each platelet in the test platelet suspension for 30 seconds in the high-speed recording described above and averaging.

As described above, image analysis may be performed on the high-speed recordings using the computer 600 to facilitate measurement of the platelet rolling speed and/or the platelet aggregation area of the test platelet suspension.

In operation 900, the flow of the test platelet suspension is stopped and the remaining volume is measured. For example, the syringe flow pump 300 may be configured to stop the flow of the test platelet suspension through the flow chamber 100. The remaining volume of platelets and/or platelet aggregates on the collagen matrix is then observed.

As described above, both the platelet aggregation area and the platelet rolling speed were measured for the platelet suspension sample and the test platelet suspension comprising the oral care composition or active ingredient sample to be evaluated. For example, camera 500 is used to record video (50fps/s, 2 minutes) of platelet behavior for two types of samples passing through system 10.

Platelet rolling speed was captured by measuring the rolling distance of each platelet during a 30 second time period (from 90 second point to 120 second point). Then, the rolling speed of the platelets is averaged to obtain the platelet rolling speed of the sample. Similarly, the total area of platelets adhered to the collagen matrix was measured at different time points (30 seconds, 60 seconds, and 120 seconds) to determine the platelet aggregation area. Because the platelet aggregation area is constantly increasing, and because the highest area is found at 120 seconds, the measurement at 120 seconds can be used as a standard statistical time point for platelet aggregation area assessment.

Examples

Aspects of the disclosure may be further understood by reference to the following examples. These examples are illustrative and are not intended to limit embodiments thereof.

Table 1 shows three oral care compositions. All ingredients in the compositions of table 1 were in the same amounts, except that test composition 1 included both oleanolic acid and eugenol, while test compositions 2 and 3 included only oleanolic acid or eugenol, respectively.

TABLE 1

As shown in example 1 below, the compositions of table 1 were evaluated in terms of platelet aggregation area and platelet rolling velocity using the in vitro platelet evaluation system and method shown in fig. 1-2. Specifically, table 2 describes the platelet aggregation area data for the compositions of table 1. Table 3 describes the platelet rolling velocity data for the compositions of table 1. As described above, the platelet aggregation area and the platelet rolling velocity can be used to determine the efficacy of the oral care composition and/or active ingredient to prevent, reduce, or stop gum bleeding.

Example 1

Platelets were obtained from healthy human donors and purified as follows: 10mL of human blood was collected in an anticoagulation tube and centrifuged at 150g for 15 minutes at room temperature. Thereafter, the upper Platelet Rich Plasma (PRP) layer was aspirated into another centrifuge tube and centrifuged at 900g for 15 minutes at room temperature. The upper Platelet Poor Plasma (PPP) layer was aspirated, and 1mL of PBS buffer was added to resuspend the deposited platelets in a 15mL centrifuge tube to produce a suspended platelet solution. The suspended platelet solution was placed under a microscope (AXIO OBSERVER A1, Zeiss AG; Jena, Germany) and platelets were counted using a hemocytometer. Then, a solution containing 3-6X 10 per ml of the compound is prepared⁵Individual platelet samples platelet suspension tubes, each with a 5mL volume of PBS.

Then, in vitro platelet evaluation was performed to measure the platelet aggregation area and the platelet rolling velocity as follows: 20 μ L of 200 μ g/mL collagen was incubated overnight (15-18 hours) at 4 ℃ on a 35mm petri dish (in the central 5 mm. times.2.5 mm area). Then, the Petri dishes were washed 3 times with 1% BSA in PBS buffer and incubated with 1% BSA in PBS for 0.5 hour at room temperature.

A flow cell system (GLYCOTECH parallel plate flow cell, GlycoTech Corp.; Gathersburg, Maryland) was set and used at 0.04mL/min (1 dyn/cm)²) Platelet suspension was flow infused for 3 minutes. Then, the flow rate was changed to or maintained at 0.04mL/min (0.1 dyn/cm)²) To reference the blood shear stress in oral capillaries, hold for 3 minutes and use a high speed camera (50fps-MIKROTRON MC1310, MIKROTRON GmbH; schlaiham (unterschlessheim, Germany) recorded the basal platelet rolling behavior (control sample) for 2 minutes. Then, the flow was stopped and the remaining volume of the platelet suspension was observed. Then, 10 μ L of the composition of table 1 was added to the platelet suspension sample. Then, at a rate of 0.32mL/min (8 dyn/cm)²) Platelet suspension samples were flow-infused for 4 minutes (total 1.2mL suspension). Then, the flow rate was changed to 0.04mL/min (0.1 dyn/cm)²) The hold time was 3 minutes, and after addition of each platelet suspension sample containing the composition sample (active sample) of table 1, platelet rolling behavior was recorded for 2 minutes using a high speed camera (50 fps).

Platelet aggregation area and platelet rolling velocity data were analyzed and tracked with IMAGE analysis software (IMAGE PRO PLUS, available from Media Cybernetics, Silver Spring, Maryland) and calculated as follows:

area of platelet aggregation

The total area of platelets adhered at different times (30 seconds, 60 seconds, 120 seconds) was measured from a 2 minute high speed recording of each platelet solution sample. Then, the amount of increase in the platelet aggregation area per sample at 120 seconds relative to the control sample was calculated using the following formula, and the three measurements per sample were averaged:

platelet rolling velocity

Platelets with a mean diameter of 2.557+/-0.929 μm were selected to measure the mean platelet rolling velocity. The rolling speed of 50 platelets was captured and calculated from each platelet solution sample labeled from 90 seconds to 120 seconds in a 2 minute high speed recording. Then, the decrease in the mean platelet rolling velocity of each platelet solution relative to the control sample was calculated using the following formula, and the three measurements for each sample were averaged:

TABLE 2

	Test composition #1	Test composition #2	Test composition #3
				Testing	7.94	7.36	7.52
Control	5.93	5.99	5.92
				Increase in mean platelet aggregation area relative to control	33.72％	22.98％	26.94％

TABLE 3

As shown in tables 2-3, the in vitro platelet evaluation systems and methods of the present invention can be used to evaluate the effect on platelet aggregation area and platelet rolling velocity of an oral care composition or active ingredient sample. For example, as shown in figures 2-3 above, test composition 1 exhibited an improved increase in mean platelet aggregation area and an improved decrease in mean platelet rolling velocity when compared to test compositions 1-2.

The in vitro platelet evaluation systems and methods of the invention can provide faster evaluation than traditional animal testing or clinical testing. For example, traditional animal testing or clinical testing requires weeks to months, while the in vitro platelet assessment systems and methods of the invention can be performed in less than 1 day, less than 6 hours, less than 4 hours, or about 2 hours or less.

The present disclosure has been described with reference to exemplary embodiments. Although a few embodiments have been shown and described, it would be appreciated by those skilled in the art that changes may be made in these embodiments without departing from the principles and spirit of the embodiments described above. It is intended that the disclosure be construed as including all such modifications and alterations insofar as they come within the scope of the appended claims or the equivalents thereof.

Claims

1. An in vitro platelet assessment method comprising:

generating a platelet suspension sample;

generating a test matrix, wherein the test matrix comprises collagen;

setting up an evaluation system comprising placing the test substrate into the evaluation system;

flowing the platelet suspension sample through the evaluation system;

adjusting the flow of the platelet suspension sample through the evaluation system;

stopping the flow of the platelet suspension sample through the evaluation system;

generating a test platelet suspension;

flowing the test platelet suspension through the evaluation system;

adjusting the flow of the test platelet suspension through the evaluation system; and

stopping the flow of the test platelet suspension through the evaluation system.

2. The method of claim 1, wherein the evaluation system comprises:

a media source that holds at least one of the platelet suspension sample and the test platelet suspension,

a flow chamber that receives at least one of the platelet suspension sample and the test platelet suspension from the media source,

a flow pump that facilitates flow of at least one of the platelet suspension sample and the test platelet suspension through the flow chamber,

a microscope configured to evaluate at least one of the platelet suspension sample and the test platelet suspension in the flow chamber, an

A camera and a computer operably connected to the microscope and configured to record images through the microscope and facilitate evaluation of at least one of the platelet suspension sample and the test platelet suspension in the flow chamber.

3. The method of claim 2, wherein the flow chamber is a parallel plate flow chamber comprising:

a bottom plate configured to control flow of at least one of the platelet suspension sample and the test platelet suspension through the flow chamber,

a sled configured to receive at least one of the platelet suspension sample and the test platelet suspension from the media source, an

A gasket configured to control a height of a flow path in the flow chamber,

wherein the base plate includes:

a flow inlet configured to be connected to the media source,

a flow outlet configured to be connected to the flow pump, an

A vacuum port configured to connect to the vacuum pump.

4. The method of claim 1, wherein the regulating the flow of the platelet suspension sample through the evaluation system comprises:

adjusting the flow rate of the platelet suspension sample simulating flow through a capillary bleeding model of the assessment system; and is

Wherein the method further comprises recording the behavior of platelets in the platelet suspension sample in the assessment system.

5. The method according to claim 4, wherein the flow rate simulated through the capillary bleeding model of the assessment system is about 0.04 mL/min.

6. The method of claim 4, wherein the behavior of platelets in the platelet suspension sample is recorded at 50fps/s for 2 minutes.

7. The method according to claim 4, wherein the behavior of platelets in the platelet suspension sample recorded in the assessment system is used to determine platelet aggregation area and platelet rolling velocity of the platelet suspension sample.

8. The method of claim 1, wherein generating a test platelet suspension comprises adding a sample of an oral care composition or active agent to the platelet suspension sample to generate the test platelet suspension.

9. The method of claim 8, wherein the regulating the flow of the test platelet suspension through the evaluation system comprises:

adjusting the flow rate of the test platelet suspension simulating flow through a capillary bleeding model of the assessment system; and is

Wherein the method further comprises recording the behavior of platelets in the test platelet suspension in the assessment system.

10. The method according to claim 9, wherein the flow rate simulated through the capillary bleeding model of the assessment system is about 0.04 mL/min.

11. The method of claim 9, wherein the behavior of platelets in the test platelet suspension is recorded at 50fps/s for 2 minutes.

12. The method of claim 9, wherein the recording of the behavior of platelets in the test platelet suspension by the evaluation system is used to determine a platelet aggregation area and a platelet rolling velocity of the test platelet suspension.

13. An in vitro platelet assessment method comprising:

generating a platelet suspension sample;

generating a test platelet suspension by adding a test agent to the platelet suspension sample;

flowing the platelet suspension sample through an evaluation system and recording the behavior of platelets in the platelet suspension sample in the evaluation system;

flowing the test platelet suspension through the evaluation system and recording the behavior of platelets in the test platelet suspension in the evaluation system;

comparing the behavior of platelets in the platelet suspension sample in the evaluation system with the behavior of platelets in the test platelet suspension in the evaluation system to determine the hemostatic efficacy of the test agent;

wherein at least one of said flowing the platelet suspension sample through the assessment system and said flowing the test platelet suspension through the assessment system comprises adjusting a flow rate that simulates a capillary bleeding model flow through the assessment system, and

wherein comparing the behavior of the platelets comprises comparing the mean platelet aggregation area and the mean platelet rolling velocity of the platelet suspension sample to the test platelet suspension.